De-ubiquitinating enzymes are a family of cysteine hydrolases which specifically cleave ubiquitin-derived substrates with the general structure Ub-L. Ub is ubiquitin(yl), L (at the C-terminus of Ub) can be any number of leaving groups ranging from small thiols and amines to Ub or other proteins. UCH-L3 is one of the members of this family of de-ubiquinating hydrolases.
Ubiquitin is a small (about 8.6 kDa) protein which is highly conserved and is especially well known for its role in targeting proteins for degradation to 26S protease. The protein has been reported to be involved in many cellular processes, inter alia cell cycle control, oncoprotein degradation, receptor function, apoptosis, regulation of transcription, stress responses, DNA repair, maintenance of chromatin structure, signaling pathways, antigen presentation and the degradation of abnormal proteins. The Ub-activating enzyme E1 is known to activate monomeric ubiquitin and forms a thioester bond with Ub C-terminus. The ligation of the Ub C-terminus to lysine side chains of acceptor proteins is then catalyzed by E2 (Ub-conjugating) and E3 (Ub ligase) enzyme families. The target proteins can be mono- or poly-ubiquitinated (the latter for example in the case of targeting for degradation by the 25S protease). Besides lysine side chains, the Ub C-terminus can also be found attached to α-amino groups in peptide bonds as all known Ub genes encode fusion proteins in which Ub is followed by a C-terminal extension.
Proteolytic processing at the Ub C-terminus is catalyzed by “DeUBiquinating enzymes” (DUBS). Numerous DUBs have been identified and fall into two families: Ubiquitin-specific Proteases (USPs) and Ubiquitin C-terminal Hydrolases (UCHs).
These enzymes all cleave the peptide bond at the C-terminus of Ub. UCH-L3 cleaves peptide extensions of up to 20 residues from Ub with high efficiency and low sequence preference. Larger folded extensions are not cleaved. Thus the UCHs are presumed to function in the regeneration of active Ub from adducts with small nucleophiles. UCH-L3 is expressed especially in hematopoietic cells. UCH-L3 is implicated in various disorders including immunological or proliferative diseases such as cancer, especially solid tumors, e.g. cancers of the colon, lung cancer or the like. The USP2 family includes a number of isoforms. UBP41 was found first in chicken, then other chicken isoforms were found (UBP46, UBP52, UBP66), and all show strong sequence similarity in the enzymatically active core region while they show differences by N- or C-terminal extension. Various ubiquitinylated test substrates have been shown to be hydrolyzed by these enzymes. DNA Sequences coding for human as well as murine USP2 homologues were identified, see Genbank. For human USP2, two isoforms have been described: a long transcript variant 1 and a short transcript variant 2. Abnormal USP function may lead to cancer and disease. For example, it has been shown that USP2a is elevated in prostate cancer cells, and inactivating the protease causes degradation of the enzyme fatty acid synthetase (which otherwise would help tumor cells to resist apoptosis and to survive under non-optimal growth conditions (see e.g. FOCUS, Apr. 16, 2004, Pat McCaffrey, News from Harvard Medical, Dental & Public Health Schools, Research Briefs, http://focus.hms.Harvard.edu/2004/April16—2004/research_briefs.html. hUBP 41 was shown to be up- or down-regulated in a number of tumor cells, e.g. breast, uterus, ovary, kidney, colon, stomach, rectum and lung tumor cells.
Proliferative diseases and other diseases that depend on deviations of regulation e.g. in signaling and/or metabolic pathways are a very common cause of death in humans and also in other warm-blooded animals.
It is therefore the problem to be solved by the present invention to make available new inhibitors that are pharmacologically advantageous and/or especially are effective via new mechanisms of action.